Project Summary/Abstract The spatial organization of chromatin critically impacts many essential genomic functions, from the regulation of gene expression to the replication of the genome. Changes in chromatin organization are associated with aging and a wide range of diseases including cancer. Despite the fundamental importance of correct chromatin organization and its effects on cellular behavior, very little is known regarding the precise folding arrangement of chromatin in various cell states, nor the molecular mechanisms that control chromatin organization in the cell nucleus. Current approaches are severely limited by a lack of experimental tools to directly trace chromatin folding, and to efficiently screen for molecular regulators of chromatin organization in the genome. To address these needs, during my postdoctoral research I invented a multiplexed DNA fluorescence in situ hybridization (FISH) technique to enable the direct three-dimensional (3D) tracing of numerous genomic regions within individual chromosomes in single cells. Here, I propose a completely new methodology ? the combination of the new FISH technique with a CRISPR/Cas9 genome-wide genetic screen ? in order to discover novel regulators of chromatin organization in a highly-efficient manner. Facilitated by these developments, I will investigate the 3D organization of chromatin in proliferative versus senescent cells, perform a genome-wide screen for unique regulators of the different chromatin organization in these cell states, and map the genetic interactions among the key regulators as well as their impact on transcription. In choosing to apply the methodology to proliferative and senescent cells, I expect to discover the genes responsible for the maintenance and regulation of chromatin organization in these specific cell states, which will be directly applicable to the many disease-relevant contexts in which they are implicated, including, importantly, aging and cancer. Ultimately, a detailed mechanistic understanding of the interplay between chromatin organization and cellular function will help to discover novel targets for diagnostic applications and new approaches for the treatment of a wide range of diseases.